Enzymes not only play a crucial role in synthesis, degradation and maintenance of numerous metabolites, but are also intimately involved in generation and degradation of biological messenger molecules that affect various pathways. One particularly significant enzyme involved in the inflammatory signaling pathway is the cyclooxygenase COX-2, also known as prostaglandin endoperoxide synthase (EC 1.14.99.1).
COX-2 is a bifunctional inducible enzyme (homodimer with a heme as cofactor) catalyzing the first committed step in the synthesis of prostaglandins, thomboxanes and other eicosanoids. There are two overall reactions catalyzed by cyclooxygenase. The first reaction is a cyclooxygenase reaction that requires two molecules of molecular oxygen. The end product of this reaction is the formation of Prostaglandin-G2. The second reaction is a peroxidase reaction that generates the final product Prostaglandin-H2.
It was hypothesized by Flower and Vane in 1972 (Nature, 240; 410-411) that more than one isozyme of cyclooxygenase may exist. Definitive proof for the existence of two isozymes (COX-1 and COX-2) of cyclooxygenase was finally shown in 1991 by Xie et al. (PNAS, 88, 2692-2692).
COX-1 is found throughout the body and it is considered constitutively expressed. It is found in abundance in the gastrointestinal track, where it produces prostaglandins (prostacyclin, PGI2, PGE2 etc.) that are considered cytoprotective. COX-1 is also found in abundance in the kidneys and in platelets.
COX-2 expression is inducible by inflammatory cytokines such as: Interleukins-1b i.e (IL)-1, and (IL)-2, Tumor Necrosis Factor-a (TNF)-a, growth Factors, and COX-2 expression is repressed by anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and Glucocorticoids. COX-2 derived prostaglandins are considered deleterious because they are found mainly at the sites of inflammation and are thought to trigger, participate in, or exacerbate the inflammation process.
COX-2 Inhibitors
It is generally believed that COX-2 specific inhibitors will reduce pain, fever, and inflammation without causing gastrointestinal or renal injury. There are two central tenets to this hypothesis: First, the prostaglandins that mediate inflammation, fever and pain are produced solely via COX-2. Second, prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1. Thus, it is thought that the toxicity of non-steroidal anti-inflammatory drugs (NSAIDS) in the GI and renal systems may be due to a lack of selectivity of those drugs with respect to inhibition of COX-1 and COX-2. Consequently, numerous scientists attempted to provide COX-2 selective inhibitors that would lead to a powerful drug without the adverse side-effects of COX-2 inhibitors that also affect COX-1, and various naturally occurring compounds and synthetic compounds were discovered with at least some degree of COX-2 specificity.
For example, curcumin, a naturally occurring compound has been reported to exhibit anti-inflammatory and analgesic activity (Satoskar R, et al. Evaluation of anti-inflammatory property of curcumin (diferuloyl methane) in patients with postoperative inflammation. lnt. J. Clin. Pharmacol. Ther. Toxicol. 1986;24(12):651-4.). Moreover, recent in vitro studies suggest inhibition of the expression and activity of COX-2 in several different gastrointestinal cell lines, including colon, esophagus and small intestine (Zhang F, et al. Curcumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells. Carcinogenesis 1999;20(3):445-51).
In another example, Thunder God vine extracts exhibited significant inhibition of COX-2 translation without any appreciable effect on COX-2 transcription or COX-2 activity. Moreover, COX-1 protein expression was unaltered by the plant extract (Lipsky P E, Tao X L. A potential new treatment for rheumatoid arthritis: thunder god vine. Semin Arthritis Rheum 1997;26(5):713-23). In a still further example, resveratol was demonstrated to reverse a phorbol ester induced increase in COX-2 mRNA and protein in human mammary and oral epithelial cells. In addition to modifying gene expression, it was also found that resveratrol directly inhibits COX-2 activity (Subbaramaiah K, et al. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 1998;273(34):21875-82).
Synthetic COX-2 inhibitors include selected salicylic acid compounds (e.g., acetyl salicylic acid, sodium salicylate, salsalate, diflunisal, salicylsalicylic acid, sulfasalazine, or olsalazine) and various para-aminophenol derivatives (e.g. acetaminophen). While such inhibitors generally are relatively strong COX-2 inhibitors, selectivity over COX-1 is frequently not as high as one would desire. Similarly, other COX-2 inhibitors including several indole and indende acetic acids (e.g. indomethacin, sulindac, etodolac), various heteroaryl acetic acids (e.g. Tolmetin, diclofenac, ketorolac), selected arylpropionic acids (e.g. ibuprofen, naproxen, flubiprofen, ketoprofen, fenoprofen, oxaprozin), and selected anthranilic acids (e.g. mefenamic acid, meclofenamic acid) will exhibit at least some undesirably high COX-1 inhibition. Further known relatively unspecific COX-2 inhibitors include various enolic acids (e.g. piroxican, tenoxicam), various pyrazolidinediones (e.g., phenylbutazone, oxyphenthratrazone), and selected alkanones (e.g. Nabumetone).
Although many of the above listed drugs are effective to at least some degree, the anti-inflammatory, analgesic, anti-fever and anti-thrombotic effect is frequently associated with in some cases substantial COX-1 inhibition. Unfortunately, COX-1 inhibition is thought to suppress various important functions, including repair and maintenance of stomach lining, which results in varying degrees of gastric ulcerations, perforations or obstructions in one-third to almost one-half of patients taking such drugs (Fries J. Toward an understanding of NSAID-related adverse events: the contribution of longitudinal data. Scand J Rheumatol Suppl 1996;102:3-8). Moreover, on the extreme end, more than 16,500 people die in the United States each year from NSAID-related gastrointestinal bleeding (Singh G. Recent considerations in nonsteroidal anti-inflammatory drug gastropathy. Am J Med 1998;105(1B)).
Recent developments finally resulted in various compounds with relatively high COX-2 specificity, and among those are Celecoxib (Celebrex; 4-(5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide) and rofecoxib (Vioxx; 3-phenyl-4-(4-(methylsulfonyl)phenyl))-2(5H)-furanone). However, while these compounds exhibit desirable IC50 values at relatively good COX-2 specificity, such compounds are typically expensive and not well tolerated in some patients, especially overt a prolonged period of administration.
Boron-containing Compounds as Enzyme Inhibitors
Boron compounds are known to show a variety of different biological activities. Very often, boron compounds show enzyme inhibitory activity, and various boron compounds have been shown to inhibit a number of enzymes alone or in a mixture with various co-inhibitors. For example, borates inhibit L-amino acid oxidase in a mixture with butanedione by interacting with the arginine residue in the active site. (Christman M F, Cardenas J M: Experientia 38 (5): 537-538 (1982)).
Similarly, borates and butanedione inhibit citrate/isocitrate-hydro-lyase, EC 4.2.1.3, again by interacting with the arginine residue from the active site. (Gawron O, Jones L: Biochem Biophys Acta 484 (2): 453-464 (1977)). Also, 2,3-butanedione or 1,2-cyclohexanedione in the presence of borates interact specifically with the guanidino group from arginine, and this fact was used for determination of arginine residues in the active site of the examined enzymes. In this way Dietl, T. and Tschesche, H. (Hoppe Seylers Z Physiol Chem: 357 (5): 657-665 (1976)) proved arginine residue is present in the active site of proteinases.
Borates (at low concentration) are also known to inhibit glyceraldehyde-3-phosphate dehydrogenase from human, pig and rabbit muscle. However, in greater concentration (above 6mM) borates inhibit esterase and acetylphosphatase activities. (Wolny M: Eur J Biochem 80 (2): 551-556 (1977)). Borates are also thought to inhibit methylation of catechol estrogen and pyrocatechol by catechol-O-methyltransferase (Beattie J H, Weersink E: J Inorg Biochem 46 (3): 153-160 (1992)).
Thus, although numerous compositions and methods for enzyme inhibition, and especially COX-2 inhibition are known in the art, all or almost all of them suffer from various disadvantages. Therefore, there is still a need to provide improved methods and compositions for enzyme inhibition, and especially for selective COX-2 inhibition.